1. Field of the Invention
The disease called Acquired Immune Deficiency Syndrome (AIDS) has been recognized only recently but has created an alarming situation in many parts of the world. The number of diagnosed cases per year has escalated rapidly and a large number of deaths has resulted. Until recently, the disease apparently has been prevalent primarly in homosexual men with multiple sexual partners, intravenous drug users, hemophiliacs, blood transfusion recipients, and close heterosexual contacts of members of the above group. Despite the mounting concern with regard to AIDS, the origin and cure of the disease have alluded the medical and scientific community although a great deal of research has already been carried out with respect to the disease.
A major advance and understanding of the basis of AIDS has been the isolation of a novel class of retrovirus from patients suffering from AIDS or AIDS-related complex (ARC). This group of viruses is variously called lymphadenopathy virus (LAV) as referred to by S. Wain Hobson, et al., Cell, 40, 9-17 (1978), human T lymphotropic virus III (HTLV-III) as described by L. Ratner, et al., Nature, 313, 277-84 (1985) and AIDS-associated retrovirus (ARV) as disclosed by R. Sanchez Pescador, et al., Science, 227, 484-492 (1985). From a morphologic and biologic standpoint, this group of viruses is most closely related to the lentivirus family.
The primary targets of affliction of the AIDS virus in the human body are specific subpopulations of T-cells. The severe immune deficiency of these patients results from an unusually low proportion of T-cells (T4) in their lymphocyte population. As a result the availability of many T4 helper functions is reduced. One of such functions is the production of antibodies by the B-cells.
The pronounced depression of cellular immunity that occurs in patients with AIDS and the quantitative modifications of subpopulations of their T-lymphocytes suggests that T-cells or a subset of T-cells might be a preferential target for the putative infectious agent. Alternatively, these modifications may result from subsequent infections. The depressed cellular immunity may result in serious opportuntistic infections in AIDS patients, many of whom develop Kaposi's Sarcoma. Multiple lymphadenopathies have also been described in homosexual males and infants who may or may not develop AIDS. The lympadenopathies may correspond to an early or milder form of the disease.
There are currently three major tests for establishing the presence of serum antibody specific for HTLV-III virus. The most widely employed procedure is the ELISA screen which uses either wells or beads coated with HTLV-III viral lysate. Specific antibody from a seropositive specimen binds to the antigen coated surface, followed by an enzyme linked, anti-human antibody and appropriate chromogenic substrate.
The second antibody detection procedure, most often used as a confirmatory test for the ELISA screen, is the enzyme-linked immunoelectrotransfer blot or Western Blot technique. An HTLV-III viral lysate is resolved by SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) into a series of bands, each defining a particular product of the viral genome. The antigen bands are then transblotted to a nitrocellulose membrane for use in a enzyme-linked immunosorbent assay procedure, similar to the ELISA screen. The principle advantage of the Western Blot is the resolution of the viral antigens into discreet bands providing greatly enhanced specificity. There are two bands elucidated by the Western Blot, either or both of which are considered diagnostic for HTLV-III specific antibody when detected by the ELISA portion of the test. The first band is the 24,000 dalton core protein. The second band is a 41,000 dalton glycoprotein which makes up a portion of the viral envelope. Equivocal results in the ELISA screen are frequently resolved by the Western Blot. Also, positive screens (especially among low risk group individuals) will often prove to be negative when reevaluated by the Western Blot.
The third antibody test is an indirect immunofluorescence assay using virus infected lymphoid cells which are reacted first with patient serum and then a fluorochrome-conjugated anti-human antibody. Fluorescence of the infected cells constitutes a positive test. The use of uninfected lymphoid cells provides a negative control which would detect antibody specific for the cells and not the virus.
The use of the ELISA screen and Western Blot confirmation tests for HTLV-III antibody has been a valuable screening tool for the elimination of contaminated blood products from the national blood supply as well as detection of infectious organ and sperm donors. However, the detection of serum antibody specific for HTLV-III virus is not a diagnosis of AIDS. Seropositivity does not prove that the patient has been exposed to live virus, is infectious, or has actively growing virus, nor does it provide any indication concerning the course of the disease. A significant percentage of seropositive hemophiliacs have failed to show the presence of virus in their blood by current techniques. Presumably, these individuals have been exposed to inactivated virus contained in the heat-treated factor VIII preparations. However, in other seropositive individuals the correlation between the presence of specific antibody and the ability to culture HTLV-III virus is strong.
The need for practical procedures for detecting the presence of antigens associated with HTLV-III viral infection in human body fluids is essential for a reliable diagnostic test for AIDS as well as for providing the means to evaluate the effect of several experimental drugs now being tested for their efficacy in treating AIDS victims.
Currently, the principle method for detecting HTLV-III virus in blood involves mixing patient lymphocytes with phytohemagglutin (PHA)-stimulated peripheral blood lymphocytes (PBLs) with the addition of fresh PHA-stimulated PPBLs every three days. Supernatant samples are then tested every three days for the presence of reverse transcriptase. Cells are evaluated by electron microscopy for the presence of virus. Clearly, this method is restricted to facilities with high level, P3 containment as well as sophisticated tissue culture and analysis capabilities. Results may take several weeks and the reliability of infecting lymphocyte cultures with virus is questionable. Use of a cell line known as H9 has helped some but most of the above problems still exist.
2. Description of the Related Art
Certain diagnostic techniques for determining the presence of AIDS or AIDS antibodies are known. One such technique is a helper-suppreser cell assay performed on T-cells and T-cell subpopulations. The reversal of the helper-suppresser ratio is taken as an indication of the possible presence of AIDS virus. This assay has low specificity and sensitivity. Another technique involves a determination of the thymosin level, the elevation of the level being an indication of the presence of AIDS. This technique gives positive results only when the disease has reached an advanced stage. Furthermore, it has been reported that the technique fails to give consistently positive reading in all cases when AIDS is known to be present. The diagnosis of AIDS in mammalian subjects involving the use of immunocyto adherents (rosette inhibition) techniques is disclosed in European Patent Application 0154499. U.S. Pat. No. 4,520,113 discloses the serological detection of antibodies to HTLV-III in sera of patients with AIDS and pre-AIDS conditions. Antigens, means and method for diagnosis of lymphadenopathy and AIDS is disclosed in European Patent Application 558109. A late differentation antigen associated with the helper inducer function of human T-cells is described in Nature (1985) 318: 465-467. The diagnosis of AIDS or lymphadenopathic syndrome using specified T-lymphotropic retrovirus antigen is discussed in EPO 138 667 A2.